Peptoid and nonpeptoid containing alpha-keto oxadiazoles as serine protease inhibitors

ABSTRACT

The present invention relates to certain substituted oxadiazole peptoids and nonpeptoids useful as inhibitors of serine proteases, especially human neutophil elastase (HNE). Compounds of the present invention are useful for the treatment or amelioration of symptoms of adult respiratory distress syndrome, septic shock, and multiple organ failure. Processes mediated by HNE are also implicated in conditions such as arthritis, periodontal disease, glomerulonephritis, and cystic fibrosis.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation application of U.S. Ser. No.09/842,543 filed Apr. 26, 2001, which in turn is a continuation-in-partof U.S. Ser. No. 09/090,046, filed Jun. 3, 1998 and U.S. Ser. No.09/090,274, filed Jun. 3, 1998, all of which are hereby incorporatedherein by reference in total.

BACKGROUND OF THE INVENTION

[0002] The serine proteases are a class of enzymes, which includeelastase, chymotrypsin, cathepsin G, trypsin and thrombin. Theseproteases have in common a catalytic triad consisting of Serine-195,Histidine-57 and Aspartic acid-102 (chymotrypsin numbering system).Human neutrophil elastase (HNE) is a proteolytic enzyme secreted bypolymorphonuclear leukocytes (PMNs) in response to a variety ofinflammatory stimuli. This release of HNE and its extracellularproteolytic activity are highly regulated and are normal, beneficialfunctions of PMNs. The degradative capacity of HNE, under normalcircumstances, is modulated by relatively high plasma concentrations ofα₁-proteinase inhibitor α₁-PI). However, stimulated PMNs produce a burstof active oxygen metabolites, some of which (hypochlorous acid forexample) are capable of oxidizing a critical methionine residue inα₁-PI. Oxidized α₁-PI has been shown to have limited potency as an HNEinhibitor and it has been proposed that alteration of thisproteinase/antiproteinase balance permits HNE to perform its degradativefunctions in localize and controlled environments.

[0003] Despite this balance of proteinase/antiproteinase activity, thereare several human disease states in which a breakdown of this controlmechanism is implicated in the pathogenesis of the condition. Impropermodulation of HNE activity has been suggested as a contributing factorin adult respiratory distress syndrome, septic shock and multiple organfailure. A series of studies also have indicated the involvement of PMNsand neutrophil elastase in myocardial ischemia-reperfusion injury.Humans with below normal levels of α₁-PI have an increased probabilityof developing emphysema. HNE-mediated processes are implicated in otherconditions such as arthritis, periodontal disease, glomerulonephritis,dermatitis, psoriasis, cystic fibrosis, chronic bronchitis,atherosclerosis, Alzheimer's disease, organ transplantation, cornealulcers, and invasion behavior of malignant tumors.

[0004] There is a need for effective inhibitors of HNE as therapeuticand as prophylactic agents for the treatment and/or prevention ofelastase-mediated problems.

SUMMARY OF THE INVENTION

[0005] In one embodiment, the present invention provides compounds offormula I

[0006] wherein

[0007] X and Y are independently O or N;

[0008] R₁ is alkyl, α,α-dialkylalkylaryl or α,α-dialkylalkyl fusedaryl-cycloalkyl wherein the cycloalkyl group is optionally substitutedwith two or more O atoms;

[0009] R₂ and R₃ are independently H or alkyl; or together form a ringconsisting of 3-5 carbons in which one or more carbon atoms of the ringcan optionally be replaced with heteroatoms selected from O, S or Nwherein N is optionally substituted with H or alkyl preferably one of R₂and R₃ is H and the other is iso-propyl; and

[0010] R₄ is alkyloxycarbonyl.

[0011] Preferably, compounds of the present invention comprise a 1,3,4oxadiazole ring (i.e., X is N; Y is O).

[0012] In one preferred embodiment of the invention, RI is alkyl, suchas tert-butyl. In another embodiment, R₁ is α,α-dialkylalkylaryl, suchas an α,α-dimethylbenzyl group. In still another preferred embodiment,R₁ is α,α-dialkylalkyl fused aryl-cycloalkyl wherein the cycloalkylgroup is substituted with two O atoms, such as anα,α-dimethyl-(3,4-methylenedioxy)benzyl group. In yet another preferredembodiment, R₂ and R₃ are independently alkyl, such as isopropyl, or H.Preferably, R₂ is isopropyl and R₃ is H.

[0013] In another embodiment, the present invention provides compoundsof formula II:

[0014] wherein

[0015] X and Y are independently O or N;

[0016] R₁, R₂, and R₃ are as above; R′₂ and R′₃ are independently H oralkyl; or together form a ring consisting of 3-5 carbon atoms in whichone or more carbon atoms of the ring can optionally be replaced byheteroatoms selected from O, S or N,

[0017] wherein

[0018] N is optionally substituted with H or alkyl;

[0019] A is a direct bond, —NH— or —OC(O)—NH—;

[0020] R₄ is H or halo; and

[0021] R₅ is H, alkyl or arylalkyl; or

[0022] a pharmaceutically acceptable salt thereof.

[0023] Preferably, compounds of this embodiment of the present inventioncomprise a 1,3,4 oxadiazole ring (i.e., X is N; Y is O).

[0024] In one preferred embodiment of the invention, R₁ is alkyl, suchas tert-butyl. In another embodiment, R₁ is α,α-dialkylalkyl fusedaryl-cycloalkyl wherein the cycloalkyl group is substituted with two Oatoms, such as an α,α-dimethyl-(3,4-methylenedioxy)benzyl group. In yetanother embodiment, R₁ is α,α-dialkylalkylaryl, such as anα,α-dimethylbenzyl group. In still another preferred embodiment, R₂ andR₃ are independently alkyl, such as isopropyl, or H. In a more preferredembodiment, R₂ is isopropyl, R₃ is H, and R₂′ and R₃′ are both H. WhereR₄ is halo, R₄ may be Cl, F, I or Br, although preferably it is F.

[0025] As used herein, the term “optionally substituted” means, whensubstituted, mono to fully substituted.

[0026] As used herein, the term “independently” means that thesubstituents may be the same or different.

[0027] As used herein, the term “alkyl” means C₁-C₁₅, and preferablyC₁-C₈. It will be understood that the alkyl group may be linear orbranched.

[0028] As used herein, the term “α,α-dialkylalkylaryl” means that thealkyl groups are substituted at the α-positions to the oxadiazole ringor to the aryl group or both. One such example is an α,α-dialkylbenzyl,wherein the a-substituents are preferably methyl, ethyl or propyl. Aspecific example is α,α-dimethylbenzyl. The term “α,α-dialkylalkyl fusedarylcycloalkyl” is defined to mean that the alkyl groups are substitutedat the α-positions to the oxadiazole ring or to the aryl group, and acycloalkyl is fused to the aryl ring. One such example of an“α,α-dialkylalkyl fused aryl-cycloalkyl” is anα,α-dialkyl-3,4-methylenedioxybenzyl group, wherein the α-substituentsare preferably methyl, ethyl or propyl; preferably they are methyl. Aspecific example includes the α,α-dimethyl-3,4-methylenedioxybenzylgroup.

[0029] As used herein, the term alkyloxycarbonyl means alkyl-O—C(O)—wherein the meaning of alkyl is defined above. One such example of analkyloxycarbonyl is methyloxycarbonyl and is defined by the formulaCH₃—O—C(O)—.

BRIEF DESCRIPTION OF THE DRAWINGS

[0030]FIG. 1 is a schematic representation of the synthetic scheme forthe Boc protected amino alcohol intermediates used in the invention.

[0031]FIG. 2 is a schematic representation of the synthetic scheme forthe compounds of one embodiment of the invention.

[0032]FIG. 3 is a schematic representation of the synthetic scheme forthe compounds of another embodiment of the invention.

DETAILED DESCRIPTION

[0033] The compounds of the present invention have been found to bepotent inhibitors of the serine protease human neutrophil elastase(HNE). They are reversible inhibitors that presumably form a transitionstate intermediate with the active site serine residue. The compoundsare characterized by their low molecular weights, high selectivity withrespect to HNE and stability regarding physiological conditions.Therefore, the compounds can be implemented to prevent, alleviate and/orotherwise treat diseases, which are mediated by the degradative effectsassociated with the presence of HNE. Their usage is of particularimportance as they relate to various human treatment in vivo but mayalso be used as a diagnostic tool in vitro.

[0034] The present invention provides, but is not limited to, specificembodiments set forth in the Examples as well as those set forth below.

[0035] The nomenclature for the embodiments is as follows (althoughembodiments disclosed indicate the stereochemistry of the 2-methylpropylgroup as having the (S)-configuration, it will be understood that boththe enantiomerically pure (R) and racemic (R,S) configurations arewithin the scope of the invention):

[0036] Example 1Methyloxycarbonyl-L-valyl-N-[1-(2-[5-(tert-butyl)-oxadiazolyl]carbonyl)-2-(S)-methylpropyl]-L-prolinamide.

[0037] Example 2Methyloxycarbonyl-L-valyl-N-[1-(2-[5-(α,α-dimethylbenzyl)-oxadiazolyl]carbonyl)-2-(S)-methylpropyl]-L-prolinamide.

[0038] Example 3Methyloxycarbonyl-L-valyl-N-[1-(2-[5-(α,α-dimethyl-3,4-methylenedioxybenzyl)-1,3,4-oxadiazolyl]carbonyl)-2-(S)-methylpropyl]-L-prolinamide.

[0039] Example 42-[6-Oxo-2-(4-fluorophenyl)-1,6-dihydro-1-pyrimidinyl]-N-[1-(2-[5-tert-butyl1,3,4-oxadiazolyl]carbonyl)-2-(S)-methylpropyl]acetamide.

[0040] Example 5 2-[5-Benzyloxycarbonylamnino-6-oxo-2-(4-fluorophenyl)1,6-dihydro-1-pyrimidinyl]-N-[1-(2-[5-(α,α-dimethyl-3,4-methylenedioxybenzyl)-1,3,4-oxadiazolyl]carbonyl)-2-(S)-methylpropyl]acetamide.

[0041] Example 62-[5-Amino-6-oxo-2-(4-fluorophenyl)-1,6-dihydro-1-pyrirridinyl]-N-[1-(2-[5-(α,α-dimethyl-3,4-methylenedioxybenzyl)-1,3,4-oxadiazolyl]carbonyl)-2-(R,S)-methylpropyl]acetamide.

[0042] Example 72-[5-Benzyloxycarbonylamino-6-oxo-2-phenyl-1,6-dihydro-1-pyrimidinyl]-N-[1-(2-[5-(α,α-dimethyl-3,4-methylenedioxybenzyl)-1,3,4-oxadiazolyl]carbonyl)-2-(S)-methylpropyl]acetamide.

[0043] Example 82-[5-Amino-6-oxo-2-phenyl-1,6-dihydro-1-pyrimidinyl]-N-[1-(2-[5-(α,α-dimethyl-3,4-methylenedioxybenzyl)-1,3,4-oxadiazolyl]carbonyl)-2-(R,S)-methylpropyl]acetamide.

[0044] Example 92-[6-Oxo-2-phenyl-1,6-dihydro-1-pyrimidinyl]-N-[1-(2-[5-(α,α-dimethyl-3,4-methylenedioxybenzyl)-1,3,4-oxadiazolyl]carbonyl)-2-(R,S)-methylpropyl]acetamnide.

[0045] Example 102-[6-Oxo-2-(4-fluorophenyl)-1,6-dihydro-1-pyrimidinyl]-N-[1-(2-[5-(α,α-dimethyl-3,4-methylenedioxybenzyl)-1,3,4-oxadiazolyl]carbonyl)-2-(R,S)-methylpropyl]acetamide.

[0046] Example 112-[6-Oxo-2-(4-fluorophenyl)-1,6-dihydro-1-pyrimidinyl]-N-[l-(2-[5-(α,α-dimethylbenzyl)-1,3,4-oxadiazolyl]carbonyl)-2-(R,S)-methylpropyl]acetamide.

[0047] Example 122-[6-Oxo-2-phenyl-1,6-dihydro-1-pyrindinyl]-N-[1-(2-[5-(α,α-dimethylbenzyl)-1,3,4-oxadiazolyl]carbonyl)-2-(R,S)-methylpropyl]acetamide.

[0048] Example 132-[5-Methyloxycarbonylamino-6-oxo-2-phenyl-1,6-dihydro-1-pyrimidinyl]-N-[1-(2-[5-(tert-butyl)-1,3,4-oxadiazolyl]carbonyl)-2-(R,S)-methylpropyl]acetamide.

[0049] The compounds of the present invention are not limited to use forinhibition of human elastase. Elastase is a member of the class ofenzymes known as serine proteases. This enzyme class also includes, forexample, chymotrypsin, cathepsin G, trypsin and thrombin. Theseproteases have in common a catalytic triad consisting of Serine-195,Histidine-57 and Aspartic acid-102 (chymotrypsin numbering system). Theprecise hydrogen bond network that exists between these amino acidresidues allows the Serine-195 hydroxyl to form a tetrahedralintermediate with the carbonyl of an amide substrate. The decompositionof this intermediate results in the release of a free amine and theacylated enzyme. In a subsequent step, this newly formed ester ishydrolyzed to give the native enzyme and the carboxylic acid. It is thiscarboxyl component that helps characterize the specificity for theenzyme. In the example in which the carboxyl component is a peptide, thealpha-substituent of the amino acid is predominately responsible for thespecificity toward the enzyme. Utilizing the accepted nomenclature bySchechter and Berger (Biochem. Biophy. Res. Commun., 27:157 (1967) andBiochem. Biophys. Res. Commun., 32:898 (1968)), the amino acid residuesin the substrate that undergo the cleavage are defined as P₁ . . . P_(n)toward the N-terminus and P₁′ . . . P_(n)′ toward the C-terminus.Therefore, the scissile bond is between the P₁ and the P₁′ residue ofthe peptide subunits. A similar nomenclature is utilized for the aminoacid residues of the enzyme that make up the binding pocketsaccommodating the subunits of the substrate, where the binding pocketfor the enzyme is designated by S₁ . . . S_(n) instead of P₁ . . . .P_(n) as for the substrate.

[0050] The characteristics for the P₁ residue defining serine proteinasespecificity is well established. The proteinases may be segregated intothree subclasses: elastases, chymases and tryptases based on thesedifferences in the P₁ residues. The elastases prefer small aliphaticmoieties such as valine whereas the chymases and tryptases prefer largearomatic hydrophobic and positively charged residues respectively.

[0051] One additional proteinase that does not fall into one of thesecategories is propyl endopeptidase. The P₁ residue defining thespecificity is a proline. This enzyme has been irnplicated in theprogression of memory loss in Alzheimer's patients. Inhibitorsconsisting of ca-keto heterocycles have recently been shown to inhibitpropyl endopeptidase (Tsutsumi et al. J. Med. Chem., 37, 3492-3502(1994)). By way of extension, α-keto heterocycles as defined hereinallow for an increased binding in P′ region of the enzyme. TABLE 1 P₁Characteristics for Proteinase Specificity Proteinase ClassRepresentative Enzyme P₁ Characteristic Elastases Human NeutrophilElastase small aliphatic residues Chymases alpha-Chymotrypsm, CathepsinG aromatic or large hydrophobic residues Tryptases Thrombin, Trypsin,Urokinase, positively charged Plasma Kallikrein, Plasminogen residuesActivator, Plasmin Other Prolyl Endopeptidase proline

[0052] Since the P₁ residue predominately defines the specificity of thesubstrate, the present invention relates to P₁-P_(n)′ modifications,specifically, certain alpha-substituted keto-heterocycles composed of1,2,4 oxadiazoles and 1,3,4-oxadiazoles. By altering thealpha-substituent to the ketone and, to some extent, the substituent onthe heterocycle, the specificity of these compounds can be directedtoward the desired proteinase (e.g., small aliphatic groups forelastase).

[0053] The efficacy of the compounds for the treatment of variousdiseases can be determined by scientific methods, which are known in theart. The following are noted as examples for HNE mediated conditions:

[0054] for acute respiratory distress syndrome, the method according tohuman neutrophil elastase (HNE) model (AARD, 141:227-677 (1990)); theendotoxin induced acute lung injury model in minipigs (AARD, 142:782-788(1990)); or the method according to human polymorphonuclearelastase-induced lung hemorrhage model in hamsters (European PatentPublication No. 0769498) may be used;

[0055] in ischemia/reperfusion, the method according to the canine modelof reperfusion injury (J. Clin. Invest., 81: 624-629 (1988)) may beused.

[0056] The compounds of the present invention, salts thereof, and theirintermediates can be prepared or manufactured as described herein or byvarious processes known to be present in the chemical art (see e.g., WO96/16080).

[0057] Alternatively, the compounds of the present invention may beprepared as described in FIGS. 1, 2 and 3. FIG. 1 relates to thesynthesis of the Boc protected amino alcohol intermediates used in theinvention. FIGS. 2 and 3 show the use of the intermediates for thesynthesis compounds of the invention.

[0058] The 2-substituted 1,3,4-oxadiazoles (3) may be prepared viaformation of methyl esters from the corresponding acids (1) utilizing,for example, thionyl chloride and methanol, followed by treatment withhydrazine in a suitable solvent to yield hydrazonic acids (2).Alternatively, esters can be prepared by methods known to one skilled inthe art or those methods described in Comprehensive OrganicTransformations (R. Larock, VCH Publishers 1989, 966-972). Reaction of(2) with triethyl orthoformate or trimethyl orthoformate and TsOH givesthe requisite 2-substituted 1,3,4-oxadiazoles (3).

[0059] Intermediate (3′) can be formed utilizing standard conditions(e.g., butyllithium, or other known alkyl lithium reagents, at lowtemperature in a polar aprotic solvent, and further, if desired,reacting with MgBr.OEt₂) and subsequently added to aldehyde (4) to givealcohol (5).

[0060] The aldehyde (4) may be prepared via any of three methods asdescribed in FIG. 1.

[0061] One method reduces the intermediate that is formed betweenBoc-Val-OH and iso-propylchloroformate with sodium borohydride to giveBoc-Valinol (12). In a subsequent step, the Boc-Valinol is oxidized withSO₃-Py in DMSO to give aldehyde (4). Another such method takes theWeinreb amide (13) that is prepared from Boc-Val-OH (11) and reduces itto the aldehyde using diisobutylaluminum hydride (DIBAL). Alternatively,one may generate the ester (14) of the amino acid followed by reductionwith DIBAL to afford aldehyde (4).

[0062] As shown in FIGS. 2 and 3, deprotection of amine (5) usinghydrochloric acid in dioxane gives the amino hydrochloride (6), which isthen coupled to the desired acid (7) or (7′) by methods available to oneskilled in the art to give intermediate (8) or (8′). Oxidation using theSwerm Oxidation, Dess-Martin's Periodinane or other methods as describedin Oxidation in Organic Chemistry (M. Hudlicky, ACS Monograph 186(1990)) yields the desired ketone (9) or (9′).

[0063] Where a compound is substituted at the 5 position of thepyrimidinone group with a benzyloxycarbonylamino group, a deprotectionstep can be conducted as described in FIG. 3. This step requires removalof the protecting group from the amine and may be carried out by anumber of methods. For example, one may utilize aluminum chloride,anisole and nitromethane in a suitable solvent such as dichloromethaneto give the 5-amino compound (10′). Other methods of deprotectionavailable in the art may also be used.

[0064] Although the compounds described herein may be administered aspure chemicals, it is preferable to present the active ingredient as apharmaceutical composition. The invention thus further provides the useof a pharmaceutical composition comprising one or more compoundstogether with one or more pharmaceutically acceptable carriers thereofand, optionally, other therapeutic and/or prophylactic ingredients. Thecarrier(s) must be ‘acceptable’ in the sense of being compatible withthe other ingredients of the composition and not deleterious to therecipient thereof.

[0065] Pharmaceutical compositions include those suitable for oral orparenteral (including intramuscular, subcutaneous and intravenous)administration. The compositions may, where appropriate, be convenientlypresented in discrete unit dosage forms and may be prepared by any ofthe methods well known in the art of pharmacy. Such methods include thestep of bringing into association the active compound with liquidcarriers, solid matrices, semi-solid carriers, finely divided solidcarriers or combination thereof, and then, if necessary, shaping theproduct into the desired delivery system.

[0066] Pharmaceutical compositions suitable for oral administration maybe presented as discrete unit dosage forms such as hard or soft gelatincapsules, cachets or tablets each containing a predetermined amount ofthe active ingredient; as a powder or as granules; as a solution, asuspension or as an emulsion. The active ingredient may also bepresented as a bolus, electuary or paste. Tablets and capsules for oraladministration may contain conventional excipients such as bindingagents, fillers, lubricants, disintegrants, or wetting agents. Thetablets may be coated according to methods well known in the art, e.g.,with enteric coatings.

[0067] Oral liquid preparations may be in the form of, for example,aqueous or oily suspension, solutions, emulsions, syrups or elixirs, ormay be presented as a dry product for constitution with water or othersuitable vehicle before use. Such liquid preparations may containconventional additives such as suspending agents, emulsifying agents,non-aqueous vehicles (which may include edible oils), or preservatives.

[0068] The compounds may also be formulated for parenteraladministration (e.g., by injection, for example, bolus injection orcontinuous infusion) and may be presented in unit dose form in ampules,pre-filled syringes, small bolus infusion containers or in multi-dosecontainers with an added preservative. The compositions may take suchforms as suspensions, solutions, or emulsions in oily or aqueousvehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents. Alternatively, the activeingredient may be in powder form, obtained by aseptic isolation ofsterile solid or by lyophilization from solution, for constitution witha suitable vehicle, e.g., sterile, pyrogen-free water, before use.

[0069] For topical administration to the epidermis, the compounds may beformulated as ointments, creams or lotions, or as the active ingredientof a transdermal patch. Suitable transdermal delivery systems aredisclosed, for example, in Fisher et al. (U.S. Pat. No. 4,788,603) orBawas et al. (U.S. Pat. Nos. 4,931,279, 4,668,504 and 4,713,224).Ointments and creams may, for example, be formulated with an aqueous oroily base with the addition of suitable thickening and/or gellingagents. Lotions may be formulated with an aqueous or oily base and willin general also contain one or more emulsifying agents, stabilizingagents, dispersing agents, suspending agents, thickening agents, orcoloring agents. The active ingredient can also be delivered viaiontophoresis, e.g., as disclosed in U.S. Pat. Nos. 4,140,122,4,383,529, or 4,051,842.

[0070] Compositions suitable for topical administration in the mouthinclude unit dosage forms such as lozenges comprising active ingredientin a flavored base, usually sucrose and acacia or tragacanth; pastillescomprising the active ingredient in an inert base such as gelatin andglycerin or sucrose and acacia; mucoadherent gels, and mouthwashescomprising the active ingredient in a suitable liquid carrier,

[0071] When desired, the above-described compositions can be adapted toprovide sustained release of the active ingredient employed, e.g., bycombination thereof with certain hydrophilic polymer matrices, e.g.,comprising natural gels, synthetic polymer gels or mixtures thereof.

[0072] The pharmaceutical compositions according to the invention mayalso contain other adjuvants such as flavorings, coloring, antimicrobialagents, or preservatives.

[0073] It will be further appreciated that the amount of the compound,or an active salt or derivative thereof, required for use in treatmentwill vary not only with the particular salt selected but also with theroute of administration, the nature of the condition being treated and,the age and condition of the patient and will be ultimately at thediscretion of the attendant physician or clinician.

[0074] In general, however, a suitable dose will be in the range of fromabout 0.5 to about 100 mg/kg/day, e.g., from about 1 to about 75 mg/kgof body weight per day, such as 3 to about 50 mg per kilogram bodyweight of the recipient per day, preferably in the range of 6 to 90mg/kg/day, most preferably in the range of 15 to 60 mg/kg/day.

[0075] The compound is conveniently administered in unit dosage form,for example, containing 0.5 to 1000 mg, conveniently 5 to 750 mg, andmost conveniently, 10 to 500 mg of active ingredient per unit dosageform.

[0076] Ideally, the active ingredient should be administered to achievepeak plasma concentrations of the active compound of from about 0.5 toabout 75 μM, more preferably, about 1 to 50 μM, and most preferably,about 2 to about 30 μM. This may be achieved, for example, by theintravenous injection of a 0.05 to 5% solution of the active ingredient,optionally in saline, or orally administered as a bolus containing about0.5-500 mg of the active ingredient. Desirable blood levels may bemaintained by continuous infusion to provide about 0.01-5.0 mg/kg/hr orby intermittent infusions containing about 0.4-15 mg/kg of the activeingredient(s).

[0077] The desired dose may be conveniently presented in a single doseor as divided doses administered at appropriate intervals, for example,as two, three, four or more sub-doses per day. The sub-dose itself maybe further divided, e.g., into a number of discrete loosely spacedadministrations, such as multiple inhalations from an insufflator or byapplication of a plurality of drops into the eye.

[0078] While the invention has been described in connection withspecific embodiments thereof, it will be understood that it is capableof further modifications and this application is intended to cover anyvariations, uses, or adaptations of the invention following, in general,the principles of the invention and including such departures from thepresent disclosure as come within known or customary practice within theart to which the invention pertains and as may be applied to theessential features hereinbefore set forth, and as follows in the scopeof the appended claims.

[0079] The following examples are given to illustrate the invention andare not intended to be inclusive in any manner.

EXAMPLES

[0080] The compounds of the present invention, salts thereof, and theirintermediates can be prepared or manufactured as described herein or byvarious processes known to be present in the chemical art. By way of anexample, the final step in the process defined here, is an oxidation ofa 2° alcohol to a ketone. As described here, this transformation from analcohol to ketone was preformed using dimethylsulfoxide and oxalylchloride followed by base, which is known as the Swern oxidation.However, modifications of the Swern oxidation are known in the art andare acceptable in this present invention. It is known that alternativeelectrophilic molecules can be substituted for oxalyl chloride such asdicyclohexylcarbodiimide, acetic anhydride, trifluoroacetic anhydride orsulfur trioxide (Mancuso et al., Synthesis 165 (1981)). Alternatively,other oxidative methods can be used such as N-chlorosuccinimide (NCS)followed by base as described by the inventors in U.S. Pat. No.5,618,792 or periodinane such as the Dess-Martin reagent. Still othermethods may also be appropriate as described in Oxidation in OrganicChemistry (M. Hudlicky, ACS Monograph 186 (1990)).

[0081] Besides the methods described below, other methods can be usedfor making substituted oxadiazole nonpeptides. U.S. Pat. No. 5,807,829,incorporated herein by reference, teaches some other methods for makingsubstituted oxadiazole nonpeptides. The skilled artisan will understandthat where a particular enantiomer is mentioned, the mirror-imageenantiomer or a mixture of enantiomers can be used.

[0082] Symbols have the standard meanings as familiar to one skilled inthe art, by way of example the following have been used: ml(milliliters), g (grams), TLC (thin layer chromatography), R_(f) (theratio of the distance moved by a compound to the distance that thesolvent front moved during the same time on a TLC plate), ¹H NMR (protonnuclear magnetic resonance), DMSO-d6 (deuterodimethylsulfoxide) andCDCl₃ (deuterochloroform).

Example 1Methyloxycarbonyl-L-valyl-N-[1-(2-[5-(tert-butyl)-oxadiazolyl]carbonyl)-2-(S)-methylpropyl]-L-prolinamide

[0083] The secondary alcohol,methyloxycarbonyl-L-valyl-N-[1-(2-[5-(tert-butyl)-oxadiazolyl]hydroxymethyl)-2-(S)-methylpropyl]-L-prolinamide,was oxidized using one of the methods known to one skilled in the art,such as, the Swem Oxidation. The intermediatemethyloxycarbonyl-L-valyl-N-[1-(2-[5-(tert-butyl)-oxadiazolyl]hydroxymethyl)-2-(S)-methylpropyl]-L-prolinamidewas prepared as follows:

[0084] A. Tert-Butylcarbohydrazonic Acid

[0085] The mixture of methyl trimethylacetate (230 ml) and hydrazinemonohydrate (170 ml) was refluxed for 24 hours. The reaction was cooledto room temperature, and concentrated under reduced pressure. Theresidue was azeotroped with toluene several times, dissolved in asaturated aqueous solution of sodium chloride, and extracted withchloroform (4×). The extract was dried over anhydrous sodium sulfate,and concentrated under reduced pressure to givetert-butylcarbohydrazonic acid (176 g) having the following physicaldata.

[0086] TLC: R_(f)=0.59, chloroform: methanol (10:1).

[0087]¹H NMR (DMSO-d₆): δ8.78 (1H, brs), 4.15 (2H, brs), 1.08 (9H, s).

[0088] B. 2-tert-Butyl-1,3,4-oxadiazole

[0089] The mixture consisting of tert-butylcarbohydrazonic acid (176 g),trimethyl orthoformate (250 ml) and p-toluenesulfonic acid monohydrate(4.3 g) was heated and methanol removed by distillation at a temperatureranging from 90° C. to 1110° C. Trimethyl orthoformate was removed (50°C./43 mm Hg) and the residue was distilled at 120° C./23 mm Hg to give2-tert-Butyl-1,3,4-oxadiazole (131 g) having the following physicaldata.

[0090] TLC: R_(f)=0.68, chloroform: methanol (10:1).

[0091]¹H NMR (DMSO-d₆): δ9.12 (1H, s), 1.36 (9H, s).

[0092] C.1-[2-(5-tert-Butyl)-1,3,4-oxadiazolyl]-2-(S)-(tert-butoxycarbonylamino)-3-methylbutan-1-ol

[0093] To a solution of 2-tert-Butyl-1,3,4-oxadiazole (62.1 g) intetrahydrofuran (1650 ml) was added n-butyllithium in hexane (1.6 M,307.8 ml) dropwise at −78° C. under an atmosphere of argon. The mixturewas stirred for 40 min at −78° C., magnesium bromide diethyl etherate(127.2 g) was added, and the resulting mixture was allowed to warm to−45° C. After 1.5 hours, a solution of2-(S)-[N-(tert-butoxycarbonyl)amino]-3-methylbutanal (90 g) intetrahydrofuran (60 ml) was added dropwise at −45° C. and allowed towarm to −15° C. The reaction mixture was quenched by addition of asaturated aqueous solution of ammonium chloride, and extracted withethyl acetate. The extract was washed with water (×3) and a saturatedaqueous solution of sodium chloride, dried over anhydrous sodium sulfateand concentrated. The residue was purified by column chromatography onsilica gel (Merck 7734) (ethyl acetate:hexane=1:20 to 1:1) to give1-[2-(5-tert-butyl)-1,3,4-oxadiazolyl]-2-(S)-(tert-butoxycarbonylamino)-3-methylbutan-1-ol(78.6 g) having the following physical data.

[0094] TLC: R_(f)=0.42, hexane:ethyl acetate (1:1).

[0095]¹H NMR (CDCl₃): δ5.16-4.90 (2H, m), 4.67 (1H, m), 4.23 (1H, m),3.90 (1H, m), 3.66 (1H, m), 1.98 (1H, m), 1.42, 1.41 and 1.36 (total18H, each s), 1.13-0.90 (6H, m).

[0096] D.1-[2-(5-tert-Butyl)-1,3,4-oxadiazolyl]-2-(S)-amino-3-methylbutan-1-olHydrochloride

[0097] To a solution of1-[2-(5-tert-butyl)-1,3,4-oxadiazolyl]-2-(S)-(tert-butoxycarbonylamino)-3-methylbutan-1-ol(76.3 g) in dioxane (200 ml) was added 4N hydrochloric acid in dioxanesolution (1000 ml) at 0° C. The reaction mixture was concentrated underreduced pressure. The residue was solidified with diethyl ether. Thesolid was azeotroped with benzene several times to give1-[2-(5-tert-butyl)-1,3,4-oxadiazolyl]-2-(S)-amino-3-methylbutan-1-olhydrochloride (66.1 g) having the following physical data.

[0098] TLC: R_(f)=0.30, chloroform:methanol (10:1);

[0099]¹H NMR (CDCl₃): δ8.50-8.10 (2H, br), 7.10-6.80 (1H, br), 5.55-5.35(1H, m),3.95-3.60 (2H, m), 2.10 (1H, m), 1.41 (9H, s), 1.20-1.00 (6H,m).

[0100] E.Methyloxycarbonyl-L-valyl-N-[1-(2-[5-(tert-butyl)-oxadiazolyl]hydroxymethyl)-2-(S)-methylpropyl]-L-prolinamide

[0101] Prepared using methyloxycarbonyl-L-Val-Pro-OH and1-[2-(5-tert-Butyl)-1,3,4-oxadiazolyl]-2-(S)-amino-3-methylbutan-1-olhydrochloride and a coupling method know to one skilled in the art.

[0102] The product had the following physical data.

[0103] TLC: R_(f)=0.58, ethyl acetate.

[0104]¹H NMR: (200 MHz, CDCl₃), 7.53 (brd., J=6.2 Hz, 1H, NH), δ5.45-5.29 (in 2H, NH, and a CH of P₁ Val), δ 4.79-4.62 (in, 1H, a CH ofPro), 4.32 (in, 1H, a CH of P₃-Val), 3.83-3.51 (in, 2H, NCH₂ of Pro),3.68 (s, 3H, CH₃O), 2.55-1.80 (in, 6H, CHs of iso-Pr, and CH₂CH₂ ofPro), 1.47 (s, 9H, CH₃s of t-Bu), 1.16-0.86 (m, 12H, CH₃s of iso-Pr).

Example 2Methyloxycarbonyl-L-valyl-N-[I-(2-[5-(α,α-dimethylbenzyl)-oxadiazolyl]carbonyl)-2-(S)-methylpropyl]-L-prolinamide

[0105] The compound was prepared by oxidizingmethyloxycarbonyl-L-valyl-N-[1-(2-[5-(α,α-dimethylbenzyl)-oxadiazolyl]hydroxymethyl)-2-(S)-methylpropyl]-L-prolinamideusing a procedure know to one skilled in the art, such as, the SwernOxidation.

[0106] The intermediate,methyloxycarbonyl-L-valyl-N-[1-(2-[5-(α,α-dimethylbenzyl)-oxadiazolyl]hydroxymethyl)-2-(S)-methylpropyl]-L-prolinamide,was prepared using methyloxycarbonyl-L-Val-Pro-OH and1-[2-(α,α-dimethylbenzyl)-1,3,4-oxadiazolyl]-2-(S)-amino-3-methylbutan-1-olhydrochloride and a coupling method know to one skilled in the art. Theintermediate1-[2-(α,α-dimethylbenzyl)-1,3,4-oxadiazolyl]-2-(S)-amino-3-methylbutan-1-olhydrochloride was prepared using a similar procedure as described inExample 11 except methyl phenylisobutyrate was used instead of methyltrimethylacetate.

[0107] The product had the following physical data.

[0108] TLC: R_(f)=0.64, ethyl acetate.

[0109] 1H NMR (200 MHz, CDCl₃): 7.84 and 7.49 (each brd., J=7.6 Hz,totally 1H, NH), 7.40-7.20 (m, 5H aromatic Hs), 5.46-5.29 (in, 2H, NHand a CH of PI Val), 4.774.60 (m, 1H, α CH of Pro), 4.404.25 (in, 1H aCH of P₃ Val), 3.84-3.55 (in, 2H, NCH₂ of Pro), 3.68 (s, 3H, CH₃O),2.55-1.76 (in, 6H, CHs of iso-Pr andCH₂CH₂ of Pro), 1.88 (s, 6H,hetC(CH₃)₂Ph), 1.12-0.82 (in, 12H, CH₃s of iso-Pr).

Example 3Methyloxycarbonyl-L-valyl-N-[1-(2-[5-(α,α-dimethyl-3,4-methylenedioxybenzyl)-1,3,4-oxadiazolyl]carbonyl)-2-(S)-methylpropyl]-L-prolinamide

[0110] The compound was prepared by oxidizingmethyloxycarbonyl-L-valyl-N-[1-(2-[5-(α,α-dimethyl-3,4-methylenedioxybenzyl)-oxadiazolyl]hydroxymethyl)-2-(S)-methylpropyl]-L-prolinamideusing a procedure know to one skilled in the art, such as, the SwernOxidation. The intermediate,methyloxycarbonyl-L-valyl-N-[1-(2-[5-(α,α-dimethyl-3,4-methylenedioxybenzyl)-oxadiazolyl]hydroxfimethyl)-2-(S)-methylpropyl]-L-prolinamide,was prepared using methyloxycarbonyl-L-Val-Pro-OH and1-[2-(α,α-dimethyl-3,4-methylenedioxybenzyl)-1,3,4-oxadiazolyl]-2-(S)-amino-3-methylbutan-1-olhydrochloride and a coupling method know to one skilled in the art. Theintermediate1-[2-(α,α-dimethyl-3,4-methylenedioxybenzyl)-1,3,4-oxadiazolyl]-2-(S)-amino-3-methylbutan-1-olhydrochloride was prepared using a similar procedure as described inExample 1 except methyl 3,4-methylenedioxyphenylisobutyrate was usedinstead of methyl trimethylacetate.

[0111] The product had the following physical data.

[0112] TLC: R_(f)=0.63, ethyl acetate.

[0113]¹H NMR (200 MHz, CDCl₃): 7.49 (d, J=6.4 Hz, 1H, NH), 6.85-6.73 (m,3H, aromatic Hs), 5.95 (s, 2H, OCH₂O), 5.46-5.28 (m, 1H a CH of Pro),4.30 (m, 1H, a CH of P₃-Val), 3.84-3.54 (m, 2H, NCH₂ of Pro), 3.68 (s,3H, CH₃O), 2.55-1.78 (m, 6H, CHs of iso-Pr, and CH₂CH₂ of Pro), 1.83 (s,6H, HetC(CH₃)2Ph), 1.11-0.85 (m, 12H, CH₃s of iso-Pr).

Example 42-[6-Oxo-2-(4-fluorophenyl)-1,6-dihydro-1-pyrimidinyl]-N-[1-(2-[5-tert-butyl-1,3,4-oxadiazolyl]carbonyl)-2-(S)-methylpropyllacetamide

[0114] To a solution of oxalyl chloride (5.80 ml) in dichloromethane(160 ml) was slowly added dropwise a solution of dimethylsulfoxide (9.44ml) in dichloromethane (16 ml) at −78° C. under an atmosphere of argon.The mixture was stirred for 30 min at 78° C. To the mixture was addeddropwise a solution of2-[6-Oxo-2-(4-fluorophenyl)-1,6-dihydro-1-pyrimidinyl]-N-[1-(2-[5-tert-butyl-1,3,4-oxadiazolyl]hydroxymethyl)-2-(S)-methylpropyl]acetamide(15.2 g) in dichloromethane (160 ml) at −78° C. The mixture was stirredfor 2 hours at −78° C. To the resulting solution was added triethylamine(97.2 ml) dropwise at −78° C. The reaction mixture was warmed up to roomtemperature, and stirred for 34 hours at the same temperature. Thereaction mixture was acidified by addition of 2N aqueous solution ofhydrochloric acid, and extracted with dichloromethane. The extract waswashed with 2N aqueous solution of hydrochloric acid, water and asaturated aqueous solution of sodium chloride, dried over anhydroussodium sulfate, and concentrated. The residue was purified by columnchromatography on silica gel using a gradient elution of 66 to 100%ethyl acetate/hexane to give2-[6-oxo-2-(4-fluorophenyl)-1,6-dihydro-1-pyrimrdinyl]-N-[1-(2-[5-tert-butyl-1,3,4-oxadiazolyl]carbonyl)-2-(S)-methylpropyl]acetamide(10.92 g) having the following physical data.

[0115] TLC: R_(f)=0.63, chloroform:methanol (10:1).

[0116]¹H NMR (CDCl₃): δ 8.00(1H, d, J=6.5 Hz), 7.64 (2H, dd, J=8.6, 5.4Hz), 7.17 (2H, t, J=8.6 Hz), 6.95 (1H, brd, J=8.4 Hz), 6.50 (1H, d,J=6.5 Hz), 5.43 (1H, dd, J=8.4, 4.8 Hz), 4.63 and 4.58 (each 1H, each d,J=15.4 Hz), 2.53 (1H, m), 1.48 (9H, s), 1.09 (3H, d, J=6.8 Hz), 0.90(3H, d, J=6.8 Hz).

[0117] The intermediate2-[6-Oxo-2-(4-fluorophenyl)-1,6-dihydro-1-pyrimidinyl]-N-[1-(2-[5-tert-butyl-1,3,4-oxadiazolyl]hydroxymethyl)-2-(S)-methylpropyl]acetamidewas prepared as follows:

[0118] A. tert-Butylcarbohydrazonic acid

[0119] t-Butylcarbohydrazonic acid was prepared as described above.

[0120] TLC: R_(f)=0.59, chloroform:methanol (10:1).

[0121]¹H NMR (DMSO-d₆): δ 8.78 (1H, brs), 4.15 (2H, brs), 1.08 (9H, s).

[0122] B. 2-tert-Butyl 1,3,4-oxadiazole

[0123] t-tert-Butyl 1,3,4-oxadizole was prepared as described above.

[0124] C.1-[2-(5-tert-Butyl)-1,3,4-oxadiazolyl]-2-(S)-(tert-butoxycarbonylamino)-3-methylbutan-1-ol

[0125] To a solution of 2-tert-Butyl-1,3,4-oxadiazole (62.1 g) intetrahydrofuran (1650 ml) was added n-butyllithium in hexane (1.6 M,307.8 ml) dropwise at −78° C. under an atmosphere of argon. The mixturewas stirred for 40 min at −78° C., magnesium bromide diethyl etherate(127.2 g) was added, and the resulting mixture was allowed to warm to−45° C. After 1.5 hours, a solution of2-(S)-[N-(tert-butoxycarbonyl)amino]-3-methylbutanal (90 g) intetrahydrofuran (60 ml) was added dropwise at −45° C. and allowed towarm to −15° C. The reaction mixture was quenched by addition of asaturated aqueous solution of ammonium chloride, and extracted withethyl acetate. The extract was washed with water (×3) and a saturatedaqueous solution of sodium chloride, dried over anhydrous sodium sulfateand concentrated. The residue was purified by column chromatography onsilica gel (Merck 7734) (ethyl acetate:hexane=1:20→1:1)

[0126] to give1-[2-(5-tert-butyl)-1,3,4-oxadiazolyl]-2-(S)-(tert-butoxycarbonylamino)-3-methylbutan-1-ol(78.6 g) having the following physical data.

[0127] TLC: R_(f)=0.42, hexane:ethyl acetate (1:1). ¹H NMR (CDCl₃): δ5.16-4.90 (2H, m), 4.67 (1H, m), 4.23 (1H, m), 3.90 (1H, m), 3.66 (1H,m), 1.98 (1H, m), 1.42, 1.41 and 1.36 (total 18H, each s), 1.13-0.90(6H, m).

[0128] D.1-[2-(5-tert-Butyl)-1,3,4-oxadiazolyl]-2-(S)-amino-3-methylbutan-1-olHydrochloride

[0129] To a solution ofI-[2-(5-tert-butyl)-1,3,4-oxadiazolyl]-2-(S)-(tert-butoxycarbonylamino)-3-methylbutan1-ol (76.3 g) in dioxane (200 ml) was added 4N hydrochloric acid indioxane solution (1000 ml) at 0° C. The reaction mixture wasconcentrated under reduced pressure. The residue was solidified withdiethyl ether. The solid was azeotroped with benzene several times togive1-[2-(5-tert-butyl)-1,3,4-oxadiazolyl]-2-(S)-amino-3-methylbutan-1-olhydrochloride (66.1 g) having the following physical data.

[0130] TLC: R_(f)=0.30, chloroform:methanol (10:1);

[0131]¹H NMR (CDCl₃): δ 8.50-8.10 (2H, br), 7.10-6.80 (1H, br),5.55-5.35 (1H, m), 3.95-3.60 (2H, m), 2.10 (1H, m), 1.41 (9H, s),1.20-1.00 (6H, m).

[0132] E.2-[6-Oxo-2-(4-fluorophenyl)-1,6-dihydro-1-pyrimidinyl]-N-[1-(2-[5-tert-butyl-1,3,4-oxadiazol]hydroxymethyl-)2-(S)-methylpropyl]acetamide

[0133] To a solution of1-[2-(5-tert-butyl)-1,3,4-oxadiazolyl]-2-(S)-amino-3-methylbutan-1-olhydrochloride (10.76 g),[6-oxo-2-(4-flurophenyl)-1,6-dihydro-1-pyrinidinyl]acetic acid (8.63 g)and 1-hydroxybenzotriazole (5.85 g) in dimethylformamide (100 ml) wasadded 1-ethyl-3-[3-(dimethylamino) propyl]carbodiimide (7.33 g) at 0° C.To the resulting mixture was added 4-methylrnorpholine (4.21 ml) at thesame temperature. The reaction mixture was stirred for 17 hours at roomtemperature. The reaction was quenched by addition of water, extractedwith ethyl acetate (×3). The extract was washed with aqueous 10% citricacid solution, a saturated aqueous solution of sodium hydrogencarbonate,water and a saturated aqueous solution of sodium chloride. The organicphase was dried over anhydrous sodium sulfate and concentrated underreduced pressure to2-[6-oxo-2-(4-fluorophenyl)-1,6-dihydro-1-pyriniidinyl]-N-[1-(2-[5-tertbutyl1,3,4-oxadiazolyl]hydroxymethyl)-2-(S)-methylpropyl]acetarnide (14.6 g)having the following physical data.

[0134] TLC: R_(f)=0.40, chloroform:methanol (10:1);

[0135]¹H NMR (DMSO-d₆): δ 8.00 and 7.94 (each 1H, each d, J=6.6 Hz),7.71 and 7.55 (each 2H, each m), 7.19 and 7.18 (each 2H, each J=6.6Hz),-6.43 and 6.38 (each 1H, each d, J=6.6 Hz), 5.13 (1H, d, J=2.2 Hz),5.05 (1H, d, J=4.4 Hz), 4.54 (2H, s), 4.43 (2H, m), 4.31 (1H, m), 4.04(1H, m), 2.20-1.52 (1H, m), 1.41 and 1.37 (each 9H, each s), 1.08, 1060,0.94 and 0.92 (each 3H, each d, J=6.6 Hz).

Example 52-[5-Benzyloxycarbonylamino-6-oxo-2-(4-fluorophenyl)-1,6-dihydro-1-pyrirnidinyl]-N-[1-(2-[5-(α,α-dimethyl-3,4-methylenedioxybenzyl)-1,3,4-oxadiazolyl]carbonyl)-2-(S)-methylpropyl]acetamide

[0136] The compound was prepared using a similar oxidative procedure asdescribed in Example 1 utilizing2-[5-benzyloxycarbonylamino-6-oxo-2-(4-fluorophenyl)-1,6-dihydro-1-pyrimidinyl]-N-[1-(2-[5-(α,α-dimethyl-3,4-methylenedioxybenzyl)-1,3,4-oxadiazolyl]hydroxymethyl)-2-(S)-methylpropyl]acetamiudefor the 2° alcohol. The title compound,2-[5-benzyloxycarbonylamino-6-oxo-2-(4-fluorophenyl)-1,6-dihydro-1-pyrimidinyl]-N-[1-(2-[5-(α,α-dimethyl-3,4-methylenedioxybenzyl)-1,3,4-oxadiazolyl]carbonyl)-2-(S)-methylpropyl]acetamide,gave the following physical data.

[0137] TLC: R_(f)=0.66, chloroform:methanol (10:1);

[0138]¹H NMR (CDCl₃): δ 8.76 (1H, brs), 7.63-7.52 (2H, m), 7.49 (1H,brs), 7.38 (5H, brs), 7.13 (2H, t, J=8.6 Hz), 6.82-6.74 (3H, m), 6.71(1H, brd, J=8.6 Hz), 5.94 (2H, s), 5.42 (1H, dd, J=8.6, 5.0 Hz), 5.22(2H, s), 4.58 (2H, brs), 2.50 (1H, m), 1.83 (6H, s), 1.05 and 0.86 (each3H, each d, J=7.0 Hz).

[0139] The intermediate2-[5-benzyloxycarbonylamino-6-oxo-2-(4-fluorophenyl)-1,6-dihydro-1-pyrimidinyl]-N-[1-(2-[5-(α,α-dirnethyl-3,4-methylenedioxybenzyl)-1,3,4-oxadiazolyl]hydroxymethyl)-2-(S)-methylpropyl]acetarnidewas prepared in an analoguous manner as described in Example 1 E using[5-benzyloxycarbonylamino-6-oxo-2-(4-fluorophenyl)-1,6-dihydro-1-pyrimidinyl]aceticacid and1-[2(α,α-dimethyl-3,4-methylenedioxybenzyl)-1,3,4-oxadiazolyl]-2-(S)-amino-3-methylbutan-1-olhydrochloride. The intermediate1-[2-(α,α-dimethyl-3,4-methylenedioxybenzyl)-1,3,4-oxadiazolyl]-2-(S)-amino-3-methylbutan-1-olhydrochloride was prepared using a similar procedure as described inExample 1 D. The heterocyclic intermediate2-(α,α-dimethyl-3,4-methylenedioxybenzyl)-1,3,4-oxadiazole gave thefollowing physical data.

[0140] TLC: R_(f)=0.69, chloroform:methanol (10:1).

[0141]¹H NMR (CDCl₃): δ 8.30 (1H, s), 6.78(1H, brs), 6.74(2H, brs), 5.94(2H, s), 1.81(6H, s).

Example 62-[5-amino-6-oxo-2-(4-fluorophenyl)-1,6-dihydro-1-pyrimidinyl]-N-[1-(2-[5-(α,α-dimethyl-3,4-methylenedioxybenzyl)-1,3,4-oxadiazolyl]carbonyl)-2-(R,S)-methylpropyl]acetamide

[0142] To2-[5-(benzyloxycarbonylamino)-6-oxo-2-(4-fluorophenyl)-1,6-dihydro-1-pyrimidinyl]-N-[1-(2-[5-(α,α-dimethyl-3,4-methylenedioxybenzyl)-1,3,4-oxadiazolyl]carbonyl)-2-(S)-methylpropyl]acetamide(1.42 g) was added 30% hydrobromic acid in acetic acid solution (50 ml).The reaction mixture was stirred for 1 hour at room temperature. Thereaction mixture was quenched by addition of ice water, extracted withethyl acetate (×2). The combined extracts were washed with water (×2)and a saturated aqueous solution of sodium chloride. The organic phasewas dried over anhydrous sodium sulfate, filtered and concentrated underreduced pressure. The residue was purified by column chromatography onsilica gel using a gradient elution of 50 to 100% ethyl acetate/hexaneto give2-[5-amino-6-oxo-2-(4-fluorophenyl)-1,6-dihydro-1-pyrimidinyl]-N-[1-(2-[5-(α,α-dimethyl-3,4-methylenedioxybenzyl)-1,3,4-oxadiazolyl]carbonyl)-2-(R,S)-methylpropyl]acetamide(457 mg) having the following physical data.

[0143] TLC: R_(f)=0.39, ethyl acetate.

[0144]¹H NMR (CDCl₃): δ 7.53 (2H, dd, J=8.8, 5.3 Hz), 7.48 (1H, s), 7.06(2H, t, J=8.8 Hz), 6.90 (1H, brd, J=8.4 Hz), 6.84-6.70 (3H, m), 5.95(2H, s), 5.43 (1H, dd, J=8.4, 4.8 Hz), 4.63 and 4.54 (each 1H Abq,J=15.0 Hz), 4.05 ((2H, brs), 2.51 (1H, m), 1.84 (6H, s), 1.06 and 0.87(each 3H, each d, J=7.0 Hz).

Example 72-[5-Benzyloxycarbonylamino-6-oxo-2-pheny-1-1,6-dihydro-1-pyrimidinyl]-N-[1-(2-[5-(α,α-dimethyl-3,4-methylenedioxybenzyl)-1,3,4-oxadiazolyl]carbonyl)-2-(R,S)-methylpropyl]acetamide

[0145] The compound was prepared using a similar oxidative procedure asdescribed in Example 1 utilizing2-[5-benzyloxycarbonylamino-6-oxo-2-phenyl-1,6-dihydro-1-pyrimidinyl]-N-[1-(2-[-5(α,α-dimethyl-3,4-methylenedioxybenzyl)-1,3,4-oxadiazolyl]hydroxymethyl)-2-(S)-methylpropyl]acetamidefor the 2° alcohol. The title compound,2-[5-benzyloxycarbonylamino-6-oxo-2-phenyl-1,6-dihydro-1-pyrimidinyl]-N-[1-(2-[5-(α,α-dimethyl-3,4-methylenedioxybenzyl)-1,3,4-oxadiazolyl]carbonyl)-2-(R,S)-methylpropyl]acetamidegave the following physical data.

[0146] TLC: R_(f)=0.34, hexane:ethyl acetate (1:1).

[0147]¹H NMR (CDCl₃): δ 8.78 1H, brs), 7.60-7.30 (1H, m), 6.78 (3H, m),6.68 (1H, d, J=8.8 Hz), 5.94 (2H, s), 5.42 (1H, dd, J=8.8, 4.8 Hz), 5.23(2H, s), 4.65 and 4.57 (2H, Abq, J=15.0 Hz), 2.49 (1H, m), 1.83 (6H, s),1.04 (3H, d, J=6.Hz), 0.84 (3H, d, J=5.8 Hz).

[0148] The intermediate2-[5-benzyloxycarbonylamino-6-oxo-2-phenyl-1,6-dihydro-1-pyrimidinyl]-N-[1-(2-[5-(α,α-dimethyl-3,4-methylenedioxybenzyl)-1,3,4-oxadiazolyl]hydroxymethyl)-2-(S)-methylpropyl]acetamidewas prepared in an analogous manner as described in Example 1 E using5-benzyloxycarbonylamino-6-oxo-2-phenyl-1,6-dihydro-1-pyrinmidinyl]aceticacid and1-[2-(α,α-dimethyl-3,4-methylenedioxybenzyl)-1,3,4-oxadiazolyl]-2-(S)-amino-3-methylbutan-1-o1 hydrochloride. The intermediate1-[2-(α,α-dimethyl-3,4-methylenedioxybenzyl)-1,3,4-oxadiazolyl]-2-(S)-amino-3-methylbutan-1-olhydrochloride was prepared using a similar procedure as described inExample 1 D. The heterocyclic intermediate2-(α,α-dimethyl-3,4-methylenedioxybenzyl)-1,3,4-oxadiazole gave thefollowing physical data.

[0149] TLC: R_(f)=0.69, chloroform: methanol (10:1).

[0150]¹H NMR (CDCl₃): δ 8.30 (1H, s), 6.78(1H, brs), 6.74(2H, brs), 5.94(2H, s), 1.81 (6H, s).

Example 82-[5-Amino-6-oxo-2-phenyl-1,6-dihydro-1-pyrimidinyl]-N-[1-(2-[5-(α,α-dimethyl-3,4-methylenedioxybenzyl)-1,3,4-oxadiazolyl]carbonyl)-2-(R,S)-methylpropyl]acetamide

[0151] The compound was prepared using a similar procedure as describedin Example 3 utilizing2-[5-benzyloxycarbonylamino-6-oxo-2-phenyl-1,6-dihydro-1-pyrimidinyl]-N-[1-(2-[5(-α,α-dimethyl-3,4-methylenedioxybenzyl)-1,3,4-oxadiazolyl]carbonyl)-2-(S)-methylpropyl]acetamide.The title compound2-[5-Amino-6-oxo-2-phenyl-1,6-dihydro-1-pyrimidinyl]-N-[1-(2-[5-(α,α-dimethyl-3,4-methylenedioxybenzyl)-1,3,4-oxadiazolyl]carbonyl)-2-(5)-methylpropyl]acetamidegave the following physical data.

[0152] TLC: R_(f)=0.40, ethyl acetate.

[0153]¹H NMR (CDCl₃): δ 7.59-7.34 (5H, m), 7.50 (1H, s), 6.86 (1H, d,J=8.2 Hz), 6.86-6.72 (3H, m), 5.95 (2H, s), 5.43 (1H, dd, J=8.2 and 4.8Hz), 4.66 1H, d, J=15.4 Hz), 4.56 (2H, f, J=15.4 hz), 4.05 (2 h, brs),2.62-2.36 (1H, m), 1.84 (6H, s), 1.05(3H, d, J=7.0 Hz), 0.85 (3H, d,J=7.0 Hz).

Example 92-[6-oxo-2-phenyl-1,6-dihydro-1-pyrimidinyl]-N-[1-(2-[5-(α,α-dimethyl-3,4-methylenedioxybenzyl)-1,3,4-oxadiazolyl]carbonyl)-2-(R,S)-methylpropyl]acetamide

[0154] TLC: R_(f)=0.46, ethyl acetate. ¹H NMR (CDCl₃): δ 8.01 (1H, d,J=6.6 Hz), 7.65-7.35 (SH, m), 6.87 (1H, d, J=8.6,Hz), 6.85-6.70 (3H, m),6.49 (1H, d, J=6.6 Hz), 5.95 (2H, s), 5.42 (1H, dd, J=8.6 and 5.0 Hz),4.67 (1H, d, J=15.2 Hz), 4.54 (1H, d, J=15.2 Hz), 2.63-2.37 (1H, m),1.84 (6H, s), 1.05 (3H, d, J=6.8 Hz), 0.85 (3H, d, J=6.8 Hz) Example 102-[6-oxo-2-(4-fluorophenyl)-1,6-dihydro-1-pyrimidinyl]-N-[1-(2-[5-(α,α-dimethyl-3,4-methylenedioxybenzyl)-1,3,4-oxadiazolyl]carbonyl)-2-(R,S)-methylpropyl]acetamide

[0155] TLC: R_(f)=0.43, ethyl acetate.

[0156]¹H NMR (CDCl₃): δ 7.99 (1H, d, J=6.6 Hz), 7.63 (2 h, dd, J=8.6,5.2 Hz), 7.14 (2H, t, J=8.6 Hz), 6.93 (1H, brd, J=8.6 Hz), 6.84-6.70(3H, m), 6.49 (1H, d, J=6.6 Hz), 5.95 (2H, s), 5.41 (1H, dd, J=8.6, 5.0Hz), 4.64 and 4.53 (each 1H, Abq, J=15.0 Hz), 2.50 (1H, m), 1.84 (6H,s), 1.06 and 0.87 (each 3H, each d, J=7.0 Hz).

Example 11 2-[6-oxo-2-(4-fluorophenyl-)1,6-dihydro-1-pyrimidinyl]-N-[1-(2-[5-(α,α-dimethylbenzyl)-1,3,4-oxadiazolyl]carbonyl)-2-(R,S)methylpropyl]acetamide

[0157] The compound was prepared using a similar oxidative procedure asdescribed in Example 1 utilizing2-[6-oxo-2-(4-fluorophenyl)-1,6-dihydro-1-pyrimidinyl]-N-[1-(2-[5-(α,α-dimethylbenzyl)-1,3,4-oxadiazolyl]hydroxymethyl)-2-(S)-methylpropyl]acetamidefor the 2° alcohol. The title compound,2-[6-oxo-2-(4-fluorophenyl)-1,6-dihydro-1-pyrimnidinyl]-N-[1-(2-[5-(α,α-dimethylbenzyl)-1,3,4-oxadiazolyl]carbonyl)-2-(S)-methylpropyl]acetamide,gave the following physical data.

[0158] TLC: R_(f)=0.42, ethyl acetate.

[0159]¹H NMR (CDCl₃): δ 7.99 (1H, d, J=6.5 Hz), 7.62 (2H, m), 7.40-7.20(5H, m), 7.14 (2H, t, J=8.8 Hz), 6.89 (1H, brd, J=8.6 Hz), 6.49 (1H, d,J=6.5 Hz), 5.42 (1H, dd, J=8.6, 5.0 Hz), 4.61 and 4.54 (each 1H, each d,J=15.0 Hz), 2.50 (1H, m), 1.88 (6H, s), 1.06 and 0.86 (each 3H, each d,J=6.7 Hz).

[0160] The intermediate2-[6-oxo-2-(4-fluorophenyl)-1,6-dihydro-1-pyrimidinyl]-N-[1-(2-[5-(α,α-dimethylbenzyl)-1,3,4-oxadiazolyl]hydroxymethyl)-2-(S)-methylpropyl]acetamidewas prepared in an analoguous manner as described in Example 1 E using[6-oxo-2-(4-fluoropheny-1)1,6-dihydro-1-pyriridinyllacetic acid and1-[2-(α,α-dimethylbenzyl)-1,3,4-oxadiazolyl]-2-(S)-amino-3-methylbutan-1-olhydrochloride. The intermediate1-[2-(α,α-dimethyl-3,4-methylenedioxybenzyl)-1,3,4-oxadiazolyl]-2-(S)-amino-3-methylbutan-1-olhydrocloride was prepared using a similar procedure as described inExample 1 D. The heterocyclic intermediate2-(α,α-dimethylbenzyl)-1,3,4-oxadiazole gave the following physicaldata.

[0161] TLC: R_(f)=0.43, ethyl acetate:hexane (1:2).

[0162]¹H NMR (CDCl₃): δ 8.31 (1H, s), 7.40-7.14 (5H, m), 1.86 (6H, s).

Example 122-[6-oxo-2-phenyl-1,6-dihydro-1-pyrimidinyl]-N-[1-(2-[5-(α,α-dimethylbenzyl)-1,3,4-oxadiazolyl]carbonyl)-2-(R,S)-methylpropyl]acetamide

[0163] The compound was prepared using a similar oxidative procedure asdescribed in Example 4 utilizing2-[6-oxo-2-phenyl-1,6-dihydro-1-pyrimidinyl]-N-[1-(2-[5-(α,α-dimethylbenzyl)-1,3,4-oxadiazolyl]hydroxymethyl)-2-(5)-methylpropyflacetamidefor the 2° alcohol. The title compound,2-[6-oxo-2-phenyl-1,6-dihydro-1-pyrimidinyl]-N-[1-(2-[5-(α,α-dimethylbenzyl)-1,3,4-oxadiazolyl]carbonyl)-2-(S)-methylpropyl]atetamide,gave the following physical data.

[0164] TLC: R_(f)=0.44, ethyl acetate.

[0165]¹H NMR (CDCl₃): δ 8.02 (1H, d, J=6.5 Hz), 7.64-7.24 (10H, m), 6.82(1H, brd, J=8.4 Hz), 6.50 (1H, d, J=6.5 Hz), 5.44 (1H, dd, J=8.4, 4.8Hz), 4.63 and 4.56 (each 1H, each d, J=15.4 Hz), 2.50 (1H, m), 1.89 (6H,s), 1.06 and 0.86 (each 3H, each d, J=6.8 Hz).

[0166] The intermediate2-[6-oxo-2-phenyl-1,6-dihydro-1-pyrimidinyl]-N-[1-(2-[5-(α,α-dimethylbenzyl)-1,3,4-oxadiazolyl]hydroxymethyl)-2-(S)-methylpropyl]acetamidewas prepared in an analoguous manner as described in Example 1 E using[6-oxo-2-phenyl-1,6-dihydro-1-pyrimidinyl]acetic acid and1-[2-(α,α-dimethylbenzyl)-1,3,4-oxadiazolyl]-2-(S)-amino-3-methylbutan-1-olhydrochloride.

Example 132-[5-Methytoxycarbonylamino-6-oxo-2-phenyl-1,6-dihydro-1-pyrinidinyl]-N-(1-(2-[5-(tert-butyl)-1,3,4-oxadiazolyl]carbonyl)-2-(R,S)methylpropyl]acetamide

[0167] Prepared by a procedure analogous to that of Example 7. Theproduct had the following physical data.

[0168] TLC: R_(f)=0.57 methanol:chlorofonm, 1:10.

[0169]¹H NMR (200 MHz, CDCl₃): 8.78 (brs, 1H, H of pyrimidinone),7.62-7.40 (m, 6H, NH and aromatic Hs), 6.73 (brd, J=8.4 Hz, 1H, CONH),5.45 (dd, J=8.4, 5.0 Hz, 1H, α CH of Val), 4.67 and 4.61 (each d, J=15.0Hz, each 1H, CH₂ of Gly), 3.81 (s, 3H, CH₃O), 2.51 (m, C) 1H, CH ofiso-Pr), 1.48 (s, 9H, CH₃s of t-Bu), 1.07 and 0.88 (each d, J=6.8 Hz,each 3H, CH3s of iso-Pr).

Example 14 In vitro Inhibition of Elastase

[0170] The following protocol was used to determine inhibitory activityof compounds described herein. The elastase used in the protocol wasderived from human sputum (HSE). A mother solution of the HSE enzyme wasprepared from commercially available HSE (875 U/mg protein, SE-563,Elastin Product Co., Inc, Missouri, USA) by diluting with saline to1,000 U/ml, which was further diluted to 2 U/ml at 0° C. prior to use.

[0171] A solution was prepared by mixing 100 μl 0.2 M HEPES-NaOH buffer(pH 8.0), 40 μl 2.5 M NaCl, 20 μl 1% polyethyleneglycol 6000, 8 μldistilled water, 10 μl of a DMSO solution of inhibitor and 2 μl solutionof N-methoxysuccinyl-Ala-Ala-Pro-Val-p-nitroaniline (at concentrationsof 100, 200 and 400 μM). The solution was incubated for 10 minutes at37° C. To this was added an enzyme solution of HSE (elastase derivedfrom human sputum). The resulting mixture was subjected to the followingrate assay.

[0172] Optical density (SPECTRA MAX 250, Molecular Devices) at 405 nmdue to p-nitroaniline generated by the enzyme reaction was measured at37° C. in order to measure the reaction rate during the period that theproduction rate of p-nitroaniline remains linear. The rate, mO.D./min.,was measured for 10 minutes at 30 second intervals immediately after theaddition of the enzyme solution. IC₅₀ values were determined bylog-logit method and converted to K, values by Dixson plot method. Thecompounds are presented in Table 2 showing the inhibition activity (K,values, nM) against HNE. TABLE 2 Biological Activity Exam- K_(I) pleName (nM) 1 Methyloxycarbonyl-L-valyl-N-[1-(2-[5-(tert-butyl)-1,3,4- 3.0oxadiazolyl]carbonyl)-2-(S)-methylpropyl]-L-prolinamide 2Methyloxycarbonyl-L-valyl-N-[1-(2-[5- 1.32(α,αdimethylbenzyl)oxadiazolyl]carbonyl)-2-(S)-methylpropyl)-L-prolinamide. 3Methyloxycarbonyl-L-valyl-N-[1-(2-[5-α,α-dimethyl-3, 0.244-methylenedioxybenzyl)-1,3,4-oxadiazolyllcarbonyl)-2-(S)-methylpropyl]-L-prolinamide. 42-[6-Oxo-2-(4-fluorophenyl)-1,6-dihydro-1-pyrimidinyl]- 44.4N-[1-(2-[5-tert-butyl-1,3,4-oxadiazolyllcarbonyl-2-(R,S)-methylpropyl]acetamide 62-[5-Amino-6-Oxo-2-(4-fluorophenyl)-1,6-dihydro-1-pyrimidinyl]-N-[1-(2-[5-(cα,α-dimethyl-3,4- 0.51methylenedioxybenzyl)-1,3,4-oxadiazolyl]carbonyl-2-(R,S)-methylpropyl]acetamide 82-[5-Amino-6-Oxo-2-phenyl-1,6-dihydro-l-pyrimidinyl]- 1.06N-[1-(2-[5-α,α-dimethyl-3,4-methylenedioxybenzyl)-1,3,4-oxadiazolyl]carbonyl-2-(R,S)-methylpropyl]acetamide 92-[6-Oxo-2-phenyl-1,6-dihydro-1-pyrimidinyl]-N-[1-(2- 0.34[5(α,α-dimethyl-3,4-methylenedioxybenzyl)-1,3,4-oxadiazolyllcarbonyl-2-(R,S)-methylpropyl]acetamide 102-[6-Oxo-2-(4-fluorophenyl)-1,6-dihydro-1-pyrimidinyl]- 1.53N-[1-(2-[5-(α,α-dimethyl-3,4-methylenedioxybenzyl)-1,3,4-oxadiazolyl]carbonyl-2-(R,S)-methylpropyl] acetamide 112-[6-Oxo-2-(4-fluorophenyl)-1,6-dihydro-1-pyrimidinyl]- 5.34N-[1-(2-[5-(α,α-dimethylbenzyl)-1,3,4-oxadiazolyl]carbonyl)-2-(R,S)-methylpropyflacetamide 122-[6-Oxo-2-phenyl-1,6-dihydro-1-pyrimidinyl]-N-[1-(2- 1.83[5-(α,α-dimethylbenzyl)-1,3,4-oxadiazolyl]carbonyl)-2-(R,S)-methylpropyl]acetamide

Example 15 Ex vivo Inhibition of Elastase

[0173] Sixty (60) minutes after the oral administration of an inhibitorwith an appropriate vehicle, a blood sample (0.9 ml) is collectedthrough the abdominal aorta by a syringe containing 0.1 ml of a 3.8%sodium citrate solution.

[0174] The blood sample is processed as follows: 60 μl of (final 0.1-1mg/ml) a suspended solution of opsonized zymosan in Hank's buffer isadded to the preincubated whole blood (540 ill) for 5 minutes at 37° C.,and the resulting mixture is incubated for 30 minutes at the sametemperature. The reaction is terminated by immersing the test tube intoice water. The reaction mixture is then centrifuged at 3,000 rpm for 10minutes at 4° C. Twenty (20) μl of each of the resulting supernatant(the Sample) is measured for elastase activity.

[0175] The mixture consisting of the following components is incubatedfor 24 hours at 37° C., and then optical density is measured at 405 nm:0.2 M tris-HCI buffer (pH 8.0) 100 μl  2.5 M NaCl 40 μl Distilled water36 μl 50 mM solution of a substrate (*)  4 μl The Sample 20 μl

[0176] A test sample mixed with 1-methyl-2-pyrrolidone instead of thesubstrate is regarded as Substrate (−). A test sample mixed with salineinstead of the Sample is regarded as Blank. The remaining elastaseactivity in the Sample is calculated according to the following:

[0177] optical density of Substrate (+)−(optical density of Substrate(−)+optical density of Blank)

[0178] as a total production of p-nitroaniline over 24 hours based on astandard curve for the amount of p-nitroaniline.

[0179] An average activity is calculated based on the test sample of 5-6animals. An agent at 3, 10 or 30 mg/kg is orally given by a forcedadministration to a 24 hour fasted animal at 60 minutes before the bloodsampling. Optical density is measured by SPECTRA MAX 250 (MolecularDevices).

[0180] Some representative results are given in Table 3. TABLE 3Representative Results of Ex-Vivo Studies Percent Inhibition atIndicated Dosage Example 3 mg/kg 10 mg/kg 30 mg/kg 2  7%  9% 62% 3 44%69% 99%

[0181]

1 1 1 4 PRT Artificial Sequence Sequence is a known commerciallyavailable substrate for elastases. 1 Ala Ala Pro Val 1

What is claimed is:
 1. A compound of formula I

wherein R₁ is selected from tert-butyl, α,α-dimethylbenzyl, andα,α-dimethyl-3,4-methylenedioxybenzyl.
 2. A compound of claim 1, to wit:methyloxycarbonyl-L-valyl-N-[1-(2-[5-α,α-dimethylbenzyl]oxadiazolyl)carbonyl-2-(S)-methylpropyl]-L-prolinamide.3. A compound of claim 1, to wit:methoxycarbonyl-L-valyl-N-[1-(2-[5-(α,α-dimethyl-3,4-methylenedioxy)benzyl]oxadiazolyl)carbonyl-2-(S)-methylpropyl]-L-prolinamide.4. A compound of claim 1, to wit:methyloxycarbonyl-L-valyl-N-[1-(2-[5-tert-butyl]oxadiazolyl)carbonyl-2-(S)-methylpropyl]-L-prolinamide.5. A compound of formula II

wherein RI is selected from tert-butyl andα,α-dimethyl-3,4-methylenedioxybenzyl.
 6. A compound of claim 5 selectedfrom:2-[5-methyloxycarbonylamino-6-oxo-2-phenyl-1,6-dihydro-1-pyrirnidinyl]-N-[1-(2-[5-tert-butyl]oxadiazolyl)carbonyl-2-(R,S)-methylpropyl]acetamide;2-[5-methyloxycarbonylamino-6-oxo-2-phenyl-1,6-dihydro-1-pyrimidinyl]-N-[1-(2-[5-tert-butyl]oxadiazolyl)carbonyl-2-(R)-methylpropyl]acetamide;or2-[5-methyloxycarbonylamino-6-oxo-2-phenyl-1,6-dihydro-1-pyrimidinyl]-N-[1-(2-[5-tert-butyl]oxadiazolyl)carbonyl-2-(S)-methylpropyl]acetamide;7. A compound of claim 5 selected from:2-[5-methyloxycarbonylamino-6-oxo-2-phenyl-1,6-dihydro-1-pyrimidinyl]-N-[1-(2-[5-(α,α-dimethyl-3,4-methylenedioxy)benzyl]oxadiazolyl)carbonyl-2-(R,S)-methylpropyl]acetamide;2-[5-methyloxycarbonylamino-6-oxo-2-phenyl-1,6-dihydro-1-pyrimidinyl]-N-[1-(2-[5-tert-butyl]oxadiazolyl)carbonyl-2-(R)-methylpropyl]acetamide;or2-[5-methyloxycarbonylamino-6-oxo-2-phenyl-1,6-dihydro-1-pyrimidinyl]-N-[1-(2-[5-tert-butyl]oxadiazolyl)carbonyl-2-(S)-methylpropyl]acetamiide.